Protein a g conjugated agarose beads

Cells were transfected with plasmid DNA for immunoprecipitation analysis and cultured in serum‐free DMEM for 24 h. The cells were lysed in lysis buffer (0.5% Triton X‐100, 150 mM NaCl, 20 mM Tris–HCl pH 7.5, 1 mM EDTA, 0.5 mM PMSF, 2 mg/ml pepstatin A, 10 mg/ml leupeptin, 5 mg/ml aprotinin). The lysates were sheared with a 21‐gauge needle, incubated on ice for 15 min, and clarified by centrifugation at 20,817 g for 15 min at 4°C. The supernatants were precleared with protein A/G‐conjugated agarose beads (Santa Cruz) and incubated with anti‐FLAG antibody covalently conjugated agarose beads (012‐22781; Fujifilm Wako); the mixtures were then rotated for a further 16 h at 4°C. The agarose beads were washed three times with wash buffer (1% Nonidet P‐40, 0.1% SDS, 0.5% deoxycholate, 150 mM NaCl, 50 mM Tris–HCl pH 7.5, 1 mM EDTA, 0.5 mM PMSF, 2 mg/ml pepstatin A, 10 mg/ml leupeptin, 5 mg/ml aprotinin) before elution with sample buffer. The immunoprecipitates were analyzed by 10% SDS–PAGE and transferred to PVDF membranes for Western blot analyses, as described previously (Miyamoto et al, 2015). Here, 15% IP fractions and 0.75% inputs were detected by Western blotting. For the validation of gene knockout, 1.5% cell lysate was applied to each lane.

After challenge, lung samples were weighed (100 mg) and homogenized in 1 ml of in non-denaturing lysis buffer containing 20 mM Tris–HCl, pH 8, 137 mM NaCl, 10% glycerol, and 1% Triton X-100, 2 mM EDTA, protease inhibitor cocktail, and phosphatase inhibitor. Homogenates were then centrifuged at 12,000 g for 20 min at 4 °C to obtain the supernatant. Equal amounts of cell or tissue extracts were incubated with anti- ERK1/2 at a dilution of 1:50 for 4 h at 4 °C in the same total volume of lysis buffer thereafter, protein A/G conjugated agarose beads (Santa Cruz Biotechnology, CA, USA) was added and incubated overnight. The agarose beads containing the immunoprecipitate was then washed with the lysis buffer five times and finally collected by centrifugation. The washed precipitate was resuspended in 30 µl kinase buffer (15 mM Tris/HCl, pH 7.2, 15 mM MgCl₂, and 1 mM dithiothreitol). ERK activity assay was analyzed using MAP kinase assay kits (Merckmilipore, Darmstadt, Germany) according to the manufacturer's instructions. The assay is based on the ability of ERK to phosphorylate the specific substrate, myelin basic protein, (MBP). The phosphorylated MBP is then analyzed by immunoblot analysis, probing with a monoclonal Phospho-specific MBP antibody.

Cell lysates were prepared in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, pH 7.6, including protease inhibitors, 1 μg/mL leupeptin, 1 μg/mL aprotinin and 1 mM phenylmethylsulfonyl fluoride), sonicated, centrifuged at 13,000 rpm for 5 min, diluted in immunoprecipitation buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.6, supplemented with the protease inhibitors as described above) and pre-cleared with 50 μL of protein A/G-conjugated agarose beads (Santa Cruz Biotechnology) for 1 h at 4°C, before incubation with corresponding antibodies. Immunoprecipitates were washed four times with immunoprecipitation buffer and re-suspended in SDS sample buffer for Western blot analysis. The concentration of running gel was 10%. After blocking, the blots were incubated with the appropriate primary antibody (1: 1,000 dilution) or Biotin-MAA/SNA (1 μg/mL). After incubation with the secondary antibody (HRP-conjugated goat anti-rabbit IgG, goat anti-mouse IgG, 1: 5,000 dilution) or Streptavidin-HRP (1: 10,000 dilution), the signal was detected with an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology, Santa Cruz, CA). Original WB films for figures are depicted in the panels of Figure S10.